Compromised blood flow autoregulation has been proposed as one of important pathological mechanisms contributing to decreased BF and retinal ganglion cell damage in glaucoma. Within this paradigm, blood flow (BF) in the eye, particularly in the optic nerve head, can no longer be held within a normal range when intraocular pressure (IOP) and/or blood pressure (BP) fluctuates. As evidenced in clinical and laboratory experiments, such hemodynamic changes result in a blood flow decrease within the ocular tissues and associated ganglion cell damage measured by retinal nerve fiber layer thickness and functional loss. Thus, early intervention targeting specific pathways leading to autoregulation dysfunction could mitigate the hemodynamic changes and protect retinal ganglion cells from damage in glaucoma. However, the current knowledge of ocular BF autoregulation associated with the mechanism of the disease is insufficient to interpret many of the phenomena observed in clinics and in experiments. Astrocytes are essential to couple neuronal activities with blood flow (known as neurovascular coupling) in both brain and retina in a feed forward mechanism. A line of studies have suggested that astrocyte is also an essential cellular element involved in blood flow autoregulation. This has clinical implications because identification of the astrocyte role in BF autoregulation is likely to lead to a novel pathological mechanism of hemodynamic change and subsequently innovative pharmacological interventions for glaucoma. This study proposes to develop new techniques to examine peri-arterial astrocyte bioactivities and vascular response in relation to intra and extravascular pressure alteration. This initiative will lead future studies by utilizing these techniques to examine hypothesized mechanisms of BF autoregulation in normal and in glaucomatous eyes. In Specific Aim 1: (a) To develop and validate a system to set and manipulate both intravascular pressure (simulating BP) and extravascular pressure (simulating IOP) to different level and, simultaneously, image calcium signals (labeled with Fluo-4AM) in peri-arterial astrocytes and vessel diameter (labeled with Dil). (b) Compare the calcium signals within the peri-arterial astrocytes and vessel diameter changes against the magnitude of mechanical stimuli induced by changes in intra- and/ or extravascular pressure in isolated pig retina. In Specific Aim 2: To develop a novel in vivo calcium imaging technique to monitor calcium changes within peri-arterial astrocytes while BP is changed. This will be achieved by intravitreal injection of a calcium indicator (Fluo-4AM). Utilizing fluorescence mode in confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography, the rat fundus is imaged continuously to monitor the calcium signals in peri-arterial astrocytes and vessel diameter, whilst the rat BP is deterministically increased and decreased. Proving the involvement of astrocytes in BF autoregulation, identification of specific targets for therapeutic intervention may protect te ocular tissue from ischemic insult and subsequent progressive RGC damage in human glaucoma.